Lyophilization-based protocol for human plasma and serum metabolomics by benchtop NMR spectroscopy: optimization and validation

NMR spectroscopy is a key technique in metabolomics, enabling the simultaneous identification and quantification of metabolites and lipoproteins in biofluids. Although high-field NMR (600 MHz) remains the gold standard, low-field or “benchtop” NMR instruments are emerging as cost-effective alternatives for translating metabolomics into clinical practice. However, their application to biofluids can be challenged by inadequate suppression of the intense water signal, which compromises spectral quality and metabolite detection. Here, lyophilization followed by reconstitution in deuterated water was investigated as a sample-preparation strategy to improve benchtop NMR analysis of commercially available human plasma and serum. The optimal lyophilization time for 1.2 mL samples was established at 18 h. A standardized reconstitution protocol using 0.7 mL of 90:10 D₂O:phosphate buffer was developed, including controlled shaking and centrifugation. WET and CPMG-WET sequences were optimized for low-field NMR acquisition, and an automated spectral-processing workflow was implemented. The proposed protocol markedly reduced the residual water signal and improved spectral quality, enabling visualization of metabolite signals otherwise poorly detectable or masked in the original samples. CPMG filtering was preferred to chemical deproteination for attenuating protein signals and improving metabolite visualization, without introducing additional sample-preparation steps. Sample integrity was evaluated at 600 MHz by comparing lyophilized/reconstituted samples with the corresponding original samples. The spectra showed substantial overlap, indicating that the overall metabolic profile was largely preserved. However, some volatile metabolites were lost, while deuteration reactions were observed for trace metabolites such as creatinine. Storage stability was evaluated in triplicate under four conditions at both 600 and 100 MHz, followed by Principal Component Analysis. Room-temperature storage showed the lowest stability, whereas −80 °C after reconstitution showed the greatest stability. Overall, this protocol markedly improved benchtop NMR spectral quality while substantially preserving the metabolic profile of plasma and serum, supporting its potential for metabolomics of blood-derived samples.